Recent Progress in Bioconversion of Lignocellulosics

Cellulase From Submerged Fermentation

51

Regardless of the assay used, the non-linearity of cellulase kinetics requires that the enzyme activity be measured based on a fixed level of conversion.

3.4

Cellulose Hydrolysis

When faced with characterizing the kinetic behavior of an enzyme or a complex of enzymes, one usually pulls out a textbook on Michaelis-Menten kinetics and applies it to the system at hand. For beta-glucosidase, which hydrolyzes the soluble substrate cellobiose to glucose, this approach is fine. Unfortunately, for cellulase enzymes producing cellobiose from cellulose, this exercise is inadequate.

The fact that cellulase enzymes act on an insoluble substrate, cellulose, moves the kinetics outside Michaelis-Menten on several counts. First of all, the enzyme can be adsorbed to the substrate or unadsorbed, but only the adsorbed enzyme acts on the cellulose. Even more puzzling is the substrate concentration. Do we count the entire substrate, or just that in close contact with the enzyme? Clearly, we have to start from first principles in characterizing the cellulase/cellulose system.

3.5

Cellulase Adsorption

The first step in cellulose hydrolysis is the adsorption of the enzyme onto the cellulose. The rate of adsorption depends on the viscosity and agitation of the system. In a dilute, well mixed system, adsorption equilibrium is established in about 5 min. The adsorption equilibrium is described empirically by a Langmuir Adsorption isotherm [31]:

Kp Pmax PL

Pads = 06 (1)

1 + Kp PL

where

Pads	adsorbed protein, mg g–1 cellulose
Pmax	saturation constant, mg g–1 cellulose
Kp	binding constant, mL mg–1
PL	protein in solution, mg mL–1

For a natural substrate like ground cellulose or fruit pulp, Pmax is of order 10 mg g–1. Chemically pretreated cellulose such as paper pulp can have much higher Pmax values. Higher dosages of cellulase than Pmax have little effect on the rate of reaction. The adsorption behavior does not follow a reversible Langmuir profile, as the enzyme is very difficult to wash off its substrate. A Langmuir isotherm implies reversible adsorption.

3.6

Enzyme Action

Adsorbed enzyme acts on its substrate. Crude cellulase contains three types of enzymatic action against cellulose: CBHI, which acts along the glucose polymer

52	J.S. Tolan · B. Foody

from the non-reducing end; CBHII, which acts along the glucose polymer from the reducing end; and endoglucanase, which acts randomly at points along the glucose polymer. There is a high degree of synergy between the action of these different cellulase enzymes.

The detailed kinetics of the individual cellulase components is a subject of research and will not be discussed here. However, some characterizations have been made by taking the cellulase complex as a whole. In this case, the behavior can be expressed as a function of the enzyme dosage by Eq. (2):

G = kErads

(2)

where

G glucose produced, mg g–1 cellulose

krate constant

Eads adsorbed enzyme concentration, mg g–1 cellulose

rreaction order

For cellulase, typically 0.15 < r < 0.7. This fractional reaction order is characteristic of many effects by cellulase, not just production of glucose. The fractional reaction order indicates a diminishing return on increasing enzyme dosage. The relationship between extent of hydrolysis and reaction time is also expressed by a fractional exponent in time, which indicates a loss of enzyme effectiveness over time.

3.7

Product Inhibition, Enzyme Inactivation, and Substrate Recalcitrance

Cellulase is inhibited by its end products, cellobiose and glucose. For Trichoderma cellulase, the end product inhibition is uncompetitive, of the form:

ri	1		(3)
4	= 0
r	1 +	I
		4
		Ki

where

r= rate of hydrolysis, (g glucose) L–1 h–1

ri	inhibited rate of hydrolysis, g L–1 h–1
I	inhibitor concentration, g L–1
Ki	inhibition constant, g L–1

For crude Trichoderma cellulase, Ki for glucose is 69 g L–1 and Ki for cellobiose is 3.3 g L–1.

Cellulase action can be inhibited or inactivated by several classes of compounds, including strong oxidants or reducing agents, metal ions, salts, solvents, and surfactants. The binding of the enzyme to cellulose can protect the enzyme from these compounds, so it is not easy to generalize the relationship between concentration and degree of inhibition (which is concentration dependent) or inactivation (which is time dependent).

Cellulase From Submerged Fermentation

53

For most cellulosic materials, there is an upper limit to the degree of conversion that can be obtained. At this point, the accessibility of the remaining cellulose to the enzyme is limited, either by its pore structure or the presence of noncellulosic components. The decrease in rate with increasing cellulose conversion is referred to as “substrate recalcitrance”.

3.8

Effect of pH and Temperature

The fact that cellulase adsorbs onto its substrates and the action has a fractional reaction order has important effects on such practical issues as the pH and temperature ranges.

If one measures the enzyme activity as a function of pH or temperature using a soluble substrate, such as hydroxyethyl cellulose, one obtains curves characteristic of many enzymes. The temperature curve follows an Arrhenius dependence at temperatures leading up to the optimum, then drops sharply at inactivating temperatures (Fig. 1). The pH curve is roughly a bell shape, with the optimum spanning 1 to 3 pH units (Fig. 2). This exercise gives a first estimate of the pH and temperature curves, but often the behavior in specific applications is quite different.

Because the enzyme adsorbs to its substrate, the nature of the substrate influences the activity profiles. For example, as the pH is varied, the charge of the substrate and particularly ionic components of the substrate will change. This can effect the enzymes activity. For this reason, pH and temperature profiles for a given enzyme can vary widely among substrates.

The fractional reaction order dependence of the extent of reaction on enzyme dosage will tend to flatten the profiles observed in actual applications. For example, if an enzyme has a reaction order of 0.5 in a given application, then a

Fig. 1. Temperature profile of Trichoderma cellulase

54	J.S. Tolan · B. Foody

Fig. 2. pH profile of Trichoderma cellulase

4¥ change in enzyme activity (caused by a change in temperature) will only cause a 2¥ difference in the amount of enzyme action. Therefore, a graph of enzyme action as a function of temperature will have a flatter profile than a graph of enzyme activity as a function of temperature.

3.9

Effect of Shear

Although not easily described at the molecular level, the presence of shear in a system will act to increase the rate of cellulase action and the extent to which cellulase can modify a substrate. Shear can involve rubbing of the substrate against itself, such as denim in a washing machine, or abrading a substrate with a non-cellulosic material, such as pumice stones, or a combination of these. Shear acts to initiate fractures in the cellulose or propagate existing fractures. The net effect of shear is to increase the extent of reaction by the enzyme, or decrease the amount of cellulase required to reach a given effect.

The bound nature of the enzyme protects it from damage from any but the most intense shear.

3.10

Beta-Glucosidase Kinetics

Unlike the major cellulase components, beta-glucosidase is a soluble enzyme acting on a soluble substrate. Beta-glucosidase is characterized by classical Michaelis-Menten kinetics, with glucose acting as a competitive inhibitor [32]:

Vmax G2

V = 009 (4)

G2 + Km 1 + 4G

Ki

Cellulase From Submerged Fermentation		55
Table 2. Beta-glucosidase Kinetic constantsa
Constant	Trichoderma	Aspergillus niger BG
	longibrachiatum BG
Vmax (g L–1 h–1) @ 0.1 IU ml–1	3.10	2.75
Km (g L–1’)	0.16	0.36
Ki (g L–1’)	0.06	0.24

a 50 °C.

where

Vreaction velocity, g L–1 h–1

Vmax maximum velocity, g L–1 h–1

G2	cellobiose concentration, g L–1
Km	Michaelis constant, g L–1

Gglucose concentration, g L–1

Ki inhibition constant, g L–1

Values of the kinetic constants for beta-glucosidase from Trichoderma and Aspergillus are shown in Table 2.

Beta-glucosidase is available within the complex mixture of crude cellulase enzymes. In addition, some microbes, including Aspergillus niger, produce betaglucosidase with little additional cellulase activity.

4

Production of Cellulase by Submerged Culture Fermentation

4.1

Production of Crude Cellulase

The term “crude cellulase” refers to the natural cellulase enzyme produced in fermentation, without genetic modification of the microbe and without downstream modifications of the enzymes. The technology for the production of crude cellulase forms the basis for cellulase production involving genetic or downstream modifications. Almost all commercial cellulase produced by submerged fermentation is made by the fungi Trichoderma, Humicola, Aspergillus, and Penicillium, and this discussion focuses on processes using these microbes.

4.2

Fermentor Operation

Cellulase manufacturers adopt specific procedures for storing and propagating cultures to obtain reproducible fermentations. The cellulase-producing fungi are typically stored frozen at –80°C or freeze-dried. To prepare the inoculum (seed) mixture, an aliquot is taken and grown in consecutive liquid cultures of increasing volume. The volume of the last step, the seed fermentor, is typically 1–10% of the main fermentor volume.

56 J.S. Tolan · B. Foody

Cellulase production is carried out in aerobic, aseptic culture, and many aspects are consistent with that of other such systems used for production of other enzymes or antibiotics. The volume of fermentors used for commercial cellulase production ranges from 95,000 liters to 285,000 liters. The fermentations are highly aerobic, and oxygen is supplied through a sparger at a rate of 0.3 to 1.2 tank volumes per minute. The fermentation vessel is designed and operated to optimize gas transfer and mixing, such as with agitators. Heat generated by the microbial metabolism and agitation is removed through a cooling jacket or coil. Baffles are placed near the wall to increase mixing efficiency and prevent vortex formation. To prevent microbial contamination, the fermentor and support equipment are sterilized before inoculation. Steam sterilization is typically carried out at 121°C for 20 min. The incoming air is filtered.

The growth medium includes defined salts, complex nutrients, surfactants, and inducer. The salts are the typical fermentation salts, including potassium phosphate, ammonium nitrate, ammonium sulfate, calcium chloride, and magnesium sulfate [33]. The complex nutrients are most often 5 to 25 g L–1 of corn steep liquor but can also include yeast extract. The surfactants are added to control or suppress foam formation. The surfactants used include commercial antifoams as well as soybean oil or palm oil. The inducers are proprietary to each manufacturer but will contain an inexpensive mixture of soluble and/or insoluble sugars. Some inducers used include milk whey, which contains lactose; Solka floc cellulose; or, sugar or paper mill waste streams.

The temperature and pH of the fermentor is monitored and controlled throughout the fermentation. For Trichoderma, the conditions are 28–30 °C, pH 4–5. For the other fungi, the temperatures and pH are higher, typically 37 °C, pH 6.2–7.

4.3

Progression of Cellulase Production

Most of the literature on the quantitative aspects of cellulase production focuses on Trichoderma, as will this discussion.

Figure 3 shows the progression of cellulase production (IU L–1 h–1) over the years 1972 to 1989. A 200-fold increase in the productivity of cellulase enzyme

Table 3. Improvement in cellulase enzyme production

Year	Technology	Cellulase Prod’n	Reference
		(IU L–1h–1)
1972	Native strain in batch culture	3	[2]
1974	Selected strain QM9414	10	[34]
1978	Strain MCG77 with continuous culture	32	[35]
1981	Selected strain RutC30	78	[36]
1982	Strain RutC30 in fed batch culture	140	[37]
1984	Strain P37 in fed batch culture	395	[38]
1989	Selected strain in continuous culture	730	[39]

Cellulase From Submerged Fermentation

57

Fig. 3. Cellulase production by Trichoderma

production by Trichoderma was achieved in this period. The steps in this improvement are listed in Table 3.

This improvement in cellulase production averaged a doubling every 2 years between 1972 and 1984 and resulted from a combination of mutation/selection of strains and the optimization of fed-batch and continuous culture.

The highest rates of productivity reported are those of Nicholson et al. [39]. The results of cellulase production in continuous culture at a dilution rate of 0.018 h–1 are summarized in Table 4. Nicholson et al. noted that the maximum cell mass maintained in the 20 liter vessel was 40 g L–1 due to foaming, but that this limitation could be overcome in larger equipment.

4.4

Fermentation Kinetics

This commentary is based on cellulase production by Trichoderma and is consistent with the general fermentation technology of Wang et al. [40].

Table 4. Cellulase production in continuous culture [39]

Parameter	85 g L–1 feed	126 g L–1 feed	180 g L–1 feed
Cell mass (g L–1)	22	33	41
Cellulase (g L–1)	22	30	40
Productivity (IU L–1 h–1)	400	570	730

58	J.S. Tolan · B. Foody

The general governing equations are as follows:

1. Growth: Cell Mass Accumulation equals Growth minus Cell Removal

dX	FoX	(5)
5	= uX – 7
dt	V

where X is the cell mass, g L–1; t is time, h; u is the specific growth rate, h–1; Fo is the volume removed, L h–1; V is the fermenter volume, L

2.Enzyme production (assumes no destruction of enzyme in the fermentor): Enzyme accumulation equals Enzyme production minus Enzyme removal

dS	Fo P	(6)
5	= qp X – 6
dt	V

where P is the enzyme concentration, g L–1; qp is the specific enzyme productivity, g (g cells)–1 h–1

3.Substrate utilization (neglect maintenance energy and substrate removal): Substrate accumulation equals Substrate Feed minus Growth minus Enzyme Production

dS	FiSo	uX	qpX	(7)
5	= 7	– 6	– 7
dt	V	Yx	Yp

where S is the substrate concentration, g L–1; Fi is the feed rate, L h–1; Yx is the yield of cell mass from substrate; Yp is the yield of protein from substrate

In addition to the governing Eqs. (5–7), specific information has been reported [37], as follows:

For Trichoderma growing and producing cellulase in a medium in which the carbon source is the limiting nutrient, 50% of the carbon is converted to CO2 by aerobic respiration, and the other 50% of the carbon is taken up as cell mass or

protein. Therefore,
Yx = 0.5	(8)
Yp = 0.5	(9)

The relation between growth rate and substrate concentration can be represented by the Monod model:

umax S

u = 0 (10)

Ks + S

where

umax	maximum growth rate, h–1
Ks	Substrate constant, g L–1

The relationship between growth rate u and specific rate of enzyme production qp is shown in Fig. 4 [37]. At the maximum growth rate, there is little protein produced. At decreasing rates of growth, qp increases, reaching a maximum

qpmax at a growth rate that is 60% of umax . Below this rate of growth, qp remains

at qpmax .

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59

Fig. 4. Enzyme productivity qp varies with dilution rate

Eqs. (5–10) and Fig. 4 are applied to the design of batch, continuous, and fed batch systems, as follows.

4.5

Batch Culture

In batch culture, Eq. (10) determines the rate of growth. For soluble substrates, during most of the culture, S Ks , and therefore u = umax . From Fig. 4, there is little protein produced at this rapid growth condition. Batch culture of readilymetabolized substrates is therefore not used to make cellulase with Trichoderma.

An interesting alternative, however, is batch culture using cellulose as the feedstock. Because cellulose will only be consumed gradually, the effective value of S is much lower than the actual cellulose concentration, which decreases the rate of cell growth u and, according to Fig. 4, increases qp . In this system, the substrate uptake rate will depend on the amount of cellulase present. Because of its complexity, the kinetics of fermentation of cellulose will not be developed further here. However, batch culture has been used for cellulase production and is an option to consider in a fermentation design, as the feedstock is inexpensive. Handling and sterilization of the insoluble solid is a concern.

4.6

Continuous Culture

In continuous culture,

Fo

4= D (11)

V

where

D = dilution rate, L h–1

60	J.S. Tolan · B. Foody

In addition,

Fi

4= D (12)

V

Combining Eqs. (11) and (5) produces the well known relation between growth rate and dilution rate at steady-state (dX/dt = 0):

u = D

(13)

Therefore, continuous culture offers the opportunity to control the rate of cell growth. From Fig. 4, this allows one to select the qp for high enzyme production.

The steady-state relation between cell mass and dilution rate is obtained by combining Eqs. (5–9):

0.5 DSo

X = 03 (14)

D + qp

The steady-state relation between protein concentration, cell mass, and dilution rate is given by combining Eqs. (6) and (11):

qpx	(15)
P = 6
D

Combining Eqs. (14) and (15) relates protein concentration and dilution rate:

So qp

P = 0.5 01 (16)

D + qp

From Eq. (16), the protein production is at a maximum at low dilution rates. This is consistent with [37] (see Fig. 5).

Fig. 5. Protein level varies with dilution rate